This invention relates to a method of sequencing peptides and proteins, wherein reagents are directed through a valve block assembly having multiple inlets and a single outlet, while reducing oxygen contamination.
Automated chemistry instrumentation has traditionally used many subsystems to accomplish fluid handling. These subsystems include valving, tubing, tubing connectors, manifolds and fluid reservoirs. Many times, the type of chemistry used in this instrumentation is sensitive to contamination from internal or external sources so it is critical to design the instrument so that contamination especially from atmospheric oxygen is eliminated or minimized. For example, in the chemistry used in protein sequencers, the so-called Edman degradation, it is extremely important to exclude oxygen from the reaction. The Edman degradation consists of two principal chemical reactions called the coupling reaction and the cleavage reaction. During the coupling reaction, phenylisothiocyanate (PITC) reacts with the protein amino groups in the presence of a base to form a phenylthiocarbamyl (PTC) derivative of the amino-terminal amino acid. The cleavage reaction results in the anilinothiazolinone (ATZ) derivative of the amino terminal amino acid being formed as it is cleaved from the protein chain. The PTC group is extremely sensitive to desulfurization by oxidation so it is imperative that the reaction be performed in the absence of oxygen. If the PTC group is oxidized, the degradation will halt because the ATZ cannot be formed.
To overcome the problem of oxygen contamination, it has become common practice to flush the reaction chamber with an inert gas such as nitrogen or argon. While this helps keep the chamber atmosphere oxygen free, it does not prevent oxygen from entering the reaction chamber as a dissolved gas in the reactants themselves. Since the chemicals used in the Edman degradation are also very corrosive, all tubing in the fluid-handling system must be chemically inert. Unfortunately, the tubing, although extremely resistant to attack by the Edman chemicals, is also very porous to the oxygen in the atmosphere and the chemicals become contaminated by the diffusing oxygen.
Previous protein sequencers have used tubing to connect remote delivery valves, pressure valves and venting valves to chemical reservoirs with the result that oxygen sensitive chemicals are directly exposed to the diffusing oxygen, not only in the tubing, but also in the reservoirs. There is a direct pathway from the atmosphere through the tubing to the contents of the reservoir from the exposed tubing. To help overcome this problem, it has been a common practice to add reducing agents to all of the chemical reservoirs. While this seems to help significantly, it is not an ideal solution to the problem. The reducing agents contribute spurious components which may interfere with the analysis of the amino acid derivative produced by the sequencer. Also, the antioxidizing effect of the reducing agents is relatively short-term. If the chemicals in the tubing are exposed for a period of days, as in the case of an idle instrument, the chemical must be replaced or the performance of the Edman degradation is greatly compromised.
Another source of oxygen contamination is leaking at the junctions between connecting tubing and other components of the fluid-handling system. Previous fluid handling systems have made extensive use of individual components connected through tubing. Every connection represents a potential source of trouble, not only from oxygen contamination but from poor performance due to variable flow rates in a leaking system. Also, since these chemicals may be corrosive or poisonous, a leak represents a hazard to operators and to the instrument itself.
Wittman-Liebold, U.S. Pat. No. 4,008,736 describes a valve arrangement in which a common conduit is formed in the valve block. All delivery valve sites lie on the same surface and are connected by zig-zagging portions of the conduit. While this type of common conduit may be cleaned by flushing, it cannot be cleaned easily with a wire. It is also difficult to machine. Reservoirs communicate with delivery valves by tubing exposed to the atmosphere.
Graffunder, U.S. Pat. No. 4,168,724 replaced the slider valves of the '736 patent with diaphragm valves. These valves are closed by fluid pressure in an actuator chamber adjacent to the diaphragm, and opened by evacuating the chamber. The zigzag sections of the common conduit intersect at the surface of the valve block. All delivery valve sites lie on the same surface.
This apparatus has several disadvantages. First, it is necessary to provide vacuum and high pressure sources. Second, the diaphragm has a tendency to cold flow into the common conduit at the point of intersection, requiring that it be moved a greater distance in order to open the valve. This in turn places greater demands on the evacuation system, and increases the wear and tear on the diaphragm. Again, reservoirs communicate with delivery valve sites by tubing.
Hunkapiller, U.S. Pat. No. 4,558,845 replaces the zigzag sections of Wittman-Liebold with straight sections which are easier to clean and to machine. However, each valve site is placed on a separate block, and the common conduit is alternately a channel in a block and tubing exposed to the atmosphere.
Hunkapiller also replaced Wittmann's actuation mechanism with a plunger that is spring-biased to a closed position. A solenoid device is used to draw the plunger to an open position. Since Hunkapiller retains the narrow access port of the previously described apparatus, his valve is likewise subject to the problem of membrane "cold flow".